Crohn's disease is an idiopathic non- caseating granulomatous disease. One of the proposed etiologies is infection with Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the causative agent of Crohn's-like disease in ruminants (Johne's disease). Recent evidences to support M paratuberculosis infection in Crohn's disease include: 1) its isolation from Crohn's disease tissues and breast milk by culture, 2) its identification in tissues by PCR assays, 3) its detection as cell wall deficient forms in tissues by in situ hybridization, 4) the long term remission (possibly cure) in an increasing number of Crohn's patients by using anti-mycobacterial therapies, and 5) by an association with the M paratuberculosis antigens p35, p36 and the 32k mycobacterial associated antigen termed HupB protein. These data suggest a causal role for mycobacteria in at least a proportion of patients with Crohn's disease. Identification of the subgroup of Crohn's disease patients infected with M paratuberculosis has been hampered due to the lack of a simple and specific serodiagnostic test to identify those who would be candidates for anti- mycobacterial therapy. The long-range objective of this proposal is to confirm M paratuberculosis p35/ p36 antigens as serologic markers and to test whether there are specific clinical/pathologic stratification(s) that correlate with their presence. We will assess the presence of M paratuberculosis infection in Crohn's disease patients by serologic testing of sera from patients and controls and in situ hybridization for the detection of the cell wall-deficient form of M paratuberculosis in involved diseased tissues. We will also use the laser capture microdissection technique to test whether M paratuberculosis are present in granulomas of Crohn's disease patients. The results from serology and molecular studies will be compared with the clinical/pathological information and demographic and epidemiologic data gathered about each patient as well as with outcome of anti-mycobacterial therapy. The results of this study should either confirm or refute the proposed etiologic association of M. paratuberculosis and Crohn's disease as well as the identification of patients with Crohn's disease and M. paratuberculosis.